Fungal Planet description sheets: 951-1041.

Novel species of fungi described in this study include those from various countries as follows: Antarctica, Apenidiella antarctica from permafrost, Cladosporium fildesense from an unidentified marine sponge. Argentina, Geastrum wrightii on humus in mixed forest. Australia, Golovinomyces glandulariae on Glandularia aristigera, Neoanungitea eucalyptorum on leaves of Eucalyptus grandis, Teratosphaeria corymbiicola on leaves of Corymbia ficifolia, Xylaria eucalypti on leaves of Eucalyptus radiata. Brazil, Bovista psammophila on soil, Fusarium awaxy on rotten stalks of Zea mays, Geastrum lanuginosum on leaf litter covered soil, Hermetothecium mikaniae-micranthae (incl. Hermetothecium gen. nov.) on Mikania micrantha, Penicillium reconvexovelosoi in soil, Stagonosporopsis vannaccii from pod of Glycine max. British Virgin Isles, Lactifluus guanensis on soil. Canada, Sorocybe oblongispora on resin of Picea rubens. Chile, Colletotrichum roseum on leaves of Lapageria rosea. China, Setophoma caverna from carbonatite in Karst cave. Colombia, Lareunionomyces eucalypticola on leaves of Eucalyptus grandis. Costa Rica, Psathyrella pivae on wood. Cyprus, Clavulina iris on calcareous substrate. France, Chromosera ambigua and Clavulina iris var. occidentalis on soil. French West Indies, Helminthosphaeria hispidissima on dead wood. Guatemala, Talaromyces guatemalensis in soil. Malaysia, Neotracylla pini (incl. Tracyllales ord. nov. and Neotracylla gen. nov.) and Vermiculariopsiella pini on needles of Pinus tecunumanii. New Zealand, Neoconiothyrium viticola on stems of Vitis vinifera, Parafenestella pittospori on Pittosporum tenuifolium, Pilidium novae-zelandiae on Phoenix sp. Pakistan, Russula quercus-floribundae on forest floor. Portugal, Trichoderma aestuarinum from saline water. Russia, Pluteus liliputianus on fallen branch of deciduous tree, Pluteus spurius on decaying deciduous wood or soil. South Africa, Alloconiothyrium encephalarti, Phyllosticta encephalarticola and Neothyrostroma encephalarti (incl. Neothyrostroma gen. nov.) on leaves of Encephalartos sp., Chalara eucalypticola on leaf spots of Eucalyptus grandis × urophylla, Clypeosphaeria oleae on leaves of Olea capensis, Cylindrocladiella postalofficium on leaf litter of Sideroxylon inerme, Cylindromonium eugeniicola (incl. Cylindromonium gen. nov.) on leaf litter of Eugenia capensis, Cyphellophora goniomatis on leaves of Gonioma kamassi, Nothodactylaria nephrolepidis (incl. Nothodactylaria gen. nov. and Nothodactylariaceae fam. nov.) on leaves of Nephrolepis exaltata, Falcocladium eucalypti and Gyrothrix eucalypti on leaves of Eucalyptus sp., Gyrothrix oleae on leaves of Olea capensis subsp. macrocarpa, Harzia metrosideri on leaf litter of Metrosideros sp., Hippopotamyces phragmitis (incl. Hippopotamyces gen. nov.) on leaves of Phragmites australis, Lectera philenopterae on Philenoptera violacea, Leptosillia mayteni on leaves of Maytenus heterophylla, Lithohypha aloicola and Neoplatysporoides aloes on leaves of Aloe sp., Millesimomyces rhoicissi (incl. Millesimomyces gen. nov.) on leaves of Rhoicissus digitata, Neodevriesia strelitziicola on leaf litter of Strelitzia nicolai, Neokirramyces syzygii (incl. Neokirramyces gen. nov.) on leaf spots of Syzygium sp., Nothoramichloridium perseae (incl. Nothoramichloridium gen. nov. and Anungitiomycetaceae fam. nov.) on leaves of Persea americana, Paramycosphaerella watsoniae on leaf spots of Watsonia sp., Penicillium cuddlyae from dog food, Podocarpomyces knysnanus (incl. Podocarpomyces gen. nov.) on leaves of Podocarpus falcatus, Pseudocercospora heteropyxidicola on leaf spots of Heteropyxis natalensis, Pseudopenidiella podocarpi, Scolecobasidium podocarpi and Ceramothyrium podocarpicola on leaves of Podocarpus latifolius, Scolecobasidium blechni on leaves of Blechnum capense, Stomiopeltis syzygii on leaves of Syzygium chordatum, Strelitziomyces knysnanus (incl. Strelitziomyces gen. nov.) on leaves of Strelitzia alba, Talaromyces clemensii from rotting wood in goldmine, Verrucocladosporium visseri on Carpobrotus edulis. Spain, Boletopsis mediterraneensis on soil, Calycina cortegadensisi on a living twig of Castanea sativa, Emmonsiellopsis tuberculata in fluvial sediments, Mollisia cortegadensis on dead attached twig of Quercus robur, Psathyrella ovispora on soil, Pseudobeltrania lauri on leaf litter of Laurus azorica, Terfezia dunensis in soil, Tuber lucentum in soil, Venturia submersa on submerged plant debris. Thailand, Cordyceps jakajanicola on cicada nymph, Cordyceps kuiburiensis on spider, Distoseptispora caricis on leaves of Carex sp., Ophiocordyceps khonkaenensis on cicada nymph. USA, Cytosporella juncicola and Davidiellomyces juncicola on culms of Juncus effusus, Monochaetia massachusettsianum from air sample, Neohelicomyces melaleucae and Periconia neobrittanica on leaves of Melaleuca styphelioides × lanceolata, Pseudocamarosporium eucalypti on leaves of Eucalyptus sp., Pseudogymnoascus lindneri from sediment in a mine, Pseudogymnoascus turneri from sediment in a railroad tunnel, Pulchroboletus sclerotiorum on soil, Zygosporium pseudomasonii on leaf of Serenoa repens. Vietnam, Boletus candidissimus and Veloporphyrellus vulpinus on soil. Morphological and culture characteristics are supported by DNA barcodes.

Individual- and county-level predictors of cervical cancer screening: a multi-level analysis.

OBJECTIVES: Despite the gains in cervical cancer screening, there remain persistent socio-economic, geographical, racial, and ethnic disparities. This study examines the combined effect of individual- and county-level characteristics on the use of cervical cancer screening tests such as Papanicolaou (Pap) tests in Texas. STUDY DESIGN: Cross-sectional study. METHODS: Individual-level information was obtained from 2014-2015 Texas Behavioral Risk Factor Surveillance System (BRFSS). Using the county of residence of the study population, the BRFSS data were linked to the American Community Survey (2010-2014) and the Area Health Resources File (2015). Women aged between 21 and 65 years, with no history of hysterectomy, and residing in 47 counties in Texas were included in the study (n = 4276). Multi-level logistic regression was used to assess the independent influences of individual- and county-level covariates on receipt of a Pap test in the past 3 years. RESULTS: The odds of timely Pap testing were lower among women aged greater than 50 years, single women, and those with low education and income (<$25,000). Black women who reside in counties with higher percentages of Hispanics (quartile 4) were less likely to be screened compared with black women living in counties with a low Hispanic population (adjusted odds ratio [OR] = 0.08 [95% confidence interval [CI]: 0.02-0.37]). County-level socio-economic status, although associated with timely screening in bivariate analysis, was not a significant predictor of screening after controlling for individual characteristics. CONCLUSIONS: There are significant disparities in the uptake of cervical cancer screening across Texas counties. Individual-level socio-economic disparities as well as the number of obstetric-gynecologic physicians in a county are predictors of these disparities.

New Infestation of Branched Broomrape, Orobanche ramosa (Orobanchaceae), on Black Medic, (Medicago lupulina) (Fabaceae), in Virginia.

Orobanche ramosa L. is arguably the most insidious of the major broomrapes, achlorophyllous root parasites of various row crops (4). It has a broad host range and is the most widely spread of any agronomically important broomrape. In the United States, California, Kentucky, and Texas have persistent populations. The California and Texas populations are carefully monitored. Isolated and ephemeral occurrences were reported from New Jersey and a New York greenhouse (1). In May 2006, remnants of capsules were found by a botany student in an urban area of Norfolk, VA. We visited the site during May 2007 and found a flowering population of approximately 100 plants parasitizing Medicago lupulina L. On the basis of size, color, and shape of the corolla and capsule and the branching pattern, the species was determined to be O. ramosa, part of a complex of closely related taxa that is currently undergoing revision. It differs from native broomrapes and the widely introduced O. minor because of its corolla color and branching habit (2). To our knowledge, this is the first record on M. lupulina although other species of Medicago are known hosts (L. J. Musselman, unpublished data). The site of the infestation is an approximately 230-m2 mowed area next to a carwash, suggesting that seeds could have come from trash removed from cars at the vacuuming station that vents onto the mowed area. Since the carwash is near a large naval base with a transient population, seeds could have come from anywhere in the world. This species exhibits little host specificity, and earlier studies have shown that numerous crops can be parasitized by plants grown from seed collected from broomrape on wild or ornamental hosts (3). Crops grown in the Middle Atlantic Region, which are especially susceptible to parasitism by O. ramosa, include potato (Solanum tuberosum), tobacco (Nicotiana tabacum), and tomato (Lycopersicon esculentum). Because of the broad host range and potential damage by this parasite, as well as its record of expanding distribution, agricultural workers should be aware of its presence in the Mid-Atlantic States. Voucher specimens from this infestation have been deposited at the following herbaria: ODU, NCU, and VPI ( http://sciweb.nybg.org/science2/IndexHerbariorum.asp ). At the time of this publication, the USDA APHIS has initiated a control program for this first documented population in Virginia (2). References: (1) R. Jain and C. L. Foy. Weed Technol. 3:608, 1989. (2) L. J. Musselman. Castanea 47:266, 1982. (3) L. J. Musselman and C. Parker. Econ. Bot. 36:270, 1982. (4) D. L. Nickrent and L. J. Musselman. The Plant Health Instructor. Online publication. doi: 10.1094/PHI-I-2004-0330-01, 2004.

Pesticide safety training among farmworker adolescents from Starr County, Texas.

This study of adolescent farmworkers describes employer compliance with pesticide safety training, a requirement of the EPA-mandated Worker Protection Standard (WPS), and identifies variables associated with having received training within the prior five years. Data are from "A Study of Work Injuries in Farmworker Children, " a three-year cohort study of high school students living along the Texas-Mexico border in Starr County. Data were collected using a web-based, self-administered, confidential survey. Of 324 students who participated in field work between January 1 and September 30, 2003, 68 (21.0%) reported ever receiving pesticide safety training. Overall, the 61 (18.8%) students who reported training within the prior five years also reported that their most recent instruction covered at least three key WPS areas (i.e., entry into a recently treated field, pesticide-related injuries/illnesses, and emergency care for pesticide exposure). Based on a multiple logistic regression, students who were male (OR = 1.97), worked only outside of Texas (OR = 2.73), worked only for commercial growers/owners (OR = 4.35), worked only for contractors (OR = 3.18), worked corn crops (OR = 2.93), and worked potato crops (OR = 3.11) were more likely to report receipt of training within the prior five years. Results suggest that increased enforcement may be needed, especially in Texas, and special educational efforts may be needed to reach female farmworker youth.

Pesticide safety training and access to field sanitation among migrant farmworker mothers from Starr County, Texas.

Very little published research describes employer compliance with EPA-mandated Worker Protection Standard (WPS) pesticide safety training and the OSHA Field Sanitation Standard among farmworker women in general and mothers specifically. A goal of both standards is limiting farmworkers' exposure to potentially hazardous agricultural pesticides. Data from a NIOSH-supported cohort study ("Injury and Illness Surveillance in Migrant Farmworker Families") allowed for examining these issues. The cohort included 267 migrant farmworker families who usually reside along the Texas-Mexico border (Starr County, Texas). Data were collected in Starr County during in-home interviews. Of 102 mothers who participated in migrant farm work during summer 2001, 57 (55.9%) reported having ever received training/instruction in the safe use of pesticides, while 47 (46.1%) reported having received training within the previous five years, as required by WPS. Of trained mothers, 91.5% to 93.6% reported that their training covered key WPS areas: (1) entry into a recently treated field, (2) pesticide related injuries/illnesses, and (3) where to go and who to contact for emergency care following exposure. Regarding access to field sanitation, 67.5% to 84.2% of 77 mothers who worked outside Texas reported employer-provided decontamination supplies (e.g., soap, wash water, towels, and toilet facilities). However, a strikingly smaller proportion (12% to 28%) of 25 mothers who worked within Texas reported access to the same resources, suggesting discrepancies in compliance across the U.S. Due to the low level of employer compliance with both WPS and OSHA mandated standards, increased enforcement and an alternate delivery of pesticide training is recommended.

Characterization of BphF, a Rieske-type ferredoxin with a low reduction potential.

BphF is a small, soluble, Rieske-type ferredoxin involved in the microbial degradation of biphenyl. The rapid, anaerobic purification of a heterologously expressed, his-tagged BphF yielded 15 mg of highly homogeneous recombinant protein, rcBphF, per liter of cell culture. The reduction potential of rcBphF, determined using a highly oriented pyrolytic graphite (HOPG) electrode, was -157+/- 2 mV vs the standard hydrogen electrode (SHE) (20 mM MOPS, 80 mM KCl, and 1 mM dithiothreitol, pH 7.0, 22 degrees C). The electron paramagnetic resonance spectrum of the reduced rcBphF is typical of a Rieske cluster while the close similarity of the circular dichroic (CD) spectra of rcBphF and BedB, a homologous protein from the benzene dioxygenase system, indicates that the environment of the cluster is highly conserved in these two proteins. The reduction potential and CD spectra of rcBphF were relatively independent of pH between 5 and 10, indicating that the pK(a)s of the cluster's histidinyl ligands are not within this range. Gel filtration studies demonstrated that rcBphF readily oligomerizes in solution. Crystals of rcBphF were obtained using sodium formate or poly(ethylene glycol) (PEG) as the major precipitant. Analysis of the intermolecular contacts in the crystal revealed a head-to-tail interaction that occludes the cluster, but is very unlikely to be found in solution. Oligomerization of rcBphF in solution was reversed by the addition of dithiothreitol and is unrelated to the noncovalent crystallographic interactions. Moreover, the oligomerization state of rcBphF did not influence the latter's reduction potential. These results indicate that the 450 mV spread in reduction potential of Rieske clusters of dioxygenase-associated ferredoxins and mitochondrial bc(1) complexes is not due to significant differences in their solvent exposure.

Balance assessment in patients with peripheral arthritis: applicability and reliability of some clinical assessments.

BACKGROUND AND PURPOSE: Many individuals with peripheral arthritis blame decreased balance as a reason for limiting their physical activity. It is therefore important to assess and improve their balance. The purpose of the present study was to evaluate the applicability and the reliability of some clinical balance assessment methods for people with arthritis and various degrees of disability. METHOD: To examine the applicability and reliability of balance tests, 65, 19 and 22 patients, respectively, with peripheral arthritis participated in sub-studies investigating the applicability, inter-rater reliability and test-retest stability of the following methods: walking on a soft surface, walking backwards, walking in a figure-of-eight, the balance sub-scale of the Index of Muscle Function (IMF), the Timed Up and Go (TUG) test and the Berg balance scale. RESULTS: For patients with moderate disability walking in a figure-of-eight was found to be the most discriminative test, whereas ceiling effects were found for the Berg balance scale. Patients with severe disability were generally able to perform the TUG test and the Berg Balance Scale without ceiling effects. Inter-rater reliability was moderate to high and test-retest stability was satisfactory for all methods assessed. CONCLUSIONS: Applicable and reliable assessment methods of clinical balance were identified for individuals with moderate and severe disability, whereas more discriminative tests need to be developed for those with limited disability.

Crystallographic structure and functional interpretation of the cytoplasmic domain of erythrocyte membrane band 3.

The red blood cell membrane (RBCM) is a primary model for animal cell plasma membranes. One of its major organizing centers is the cytoplasmic domain of band 3 (cdb3), which links multiple proteins to the membrane. Included among its peripheral protein ligands are ankyrin (the major bridge to the spectrin-actin skeleton), protein 4. 1, protein 4.2, aldolase, glyceraldehyde-3-phosphate dehydrogenase, phosphofructokinase, deoxyhemoglobin, p72syk protein tyrosine kinase, and hemichromes. The crystal structure of cdb3 is reported at 0.26 nm (2.6 A) resolution. A tight symmetric dimer is formed by cdb3; it is stabilized by interlocked dimerization arms contributed by both monomers. Each subunit also includes a larger peripheral protein binding domain with an alpha(+) beta-fold. The binding sites of several peripheral proteins are localized in the structure, and the nature of the major conformational change that regulates membrane-skeletal interactions is evaluated. An improved structural definition of the protein network at the inner surface of the RBCM is now possible.

Steady-state kinetic characterization and crystallization of a polychlorinated biphenyl-transforming dioxygenase.

The oxygenase component of biphenyl dioxygenase (BPDO) from Comamonas testosteroni B-356 dihydroxylates biphenyl and some polychlorinated biphenyls (PCBs), thereby initiating their degradation. Overexpressed, anaerobically purified BPDO had a specific activity of 4.9 units/mg, and its oxygenase component appeared to contain a full complement of Fe(2)S(2) center and catalytic iron. Oxygenase crystals in space group R3 were obtained under anaerobic conditions using polyethylene glycol as the precipitant. X-ray diffraction was measured to 1.6 A. Steady-state kinetics assays demonstrated that BPDO had an apparent k(cat)/K(m) for biphenyl of (1.2 +/- 0.1) x 10(6) M(-1) s(-1) in air-saturated buffer. Moreover, BPDO transformed dichlorobiphenyls (diClBs) in the following order of apparent specificities: 3,3'- > 2,2'- > 4, 4'-diClB. Strikingly, the ability of BPDO to utilize O(2) depended strongly on the biphenyl substrate: k(cat)/K(m(O(2))) = (3.6 +/- 0. 3), (0.06 +/- 0.02), and (0.4 +/- 0.07) x 10(5) M(-1) s(-1) in the presence of biphenyl and 2,2'- and 3,3'-diClBs, respectively. Moreover, biphenyl/O(2) consumed was 0.97, 0.44, 0.63, and 0.48 in the presence of biphenyl and 2,2'-, 3,3'-, and 4,4'-diClBs, respectively. Within experimental error, the balance of consumed O(2) was detected as H(2)O(2). Thus, PCB congeners such as 2, 2'-diClB exact a high energetic cost, produce a cytotoxic compound (H(2)O(2)), and can inhibit degradation of other congeners. Each of these effects would be predicted to inhibit the aerobic microbial catabolism of PCBs.

A cluster exposed: structure of the Rieske ferredoxin from biphenyl dioxygenase and the redox properties of Rieske Fe-S proteins.

BACKGROUND: Ring-hydroxylating dioxygenases are multicomponent systems that initiate biodegradation of aromatic compounds. Many dioxygenase systems include Rieske-type ferredoxins with amino acid sequences and redox properties remarkably different from the Rieske proteins of proton-translocating respiratory and photosynthetic complexes. In the latter, the [Fe2S2] clusters lie near the protein surface, operate at potentials above +300 mV at pH 7, and express pH- and ionic strength-dependent redox behavior. The reduction potentials of the dioxygenase ferredoxins are approximately 150 mV and are pH-independent. These distinctions were predicted to arise from differences in the exposure of the cluster and/or interactions of the histidine ligands. RESULTS: The crystal structure of BphF, the Rieske-type ferredoxin associated with biphenyl dioxygenase, was determined by multiwavelength anomalous diffraction and refined at 1.6 A resolution. The structure of BphF was compared with other Rieske proteins at several levels. BphF has the same two-domain fold as other Rieske proteins, but it lacks all insertions that give the others unique structural features. The BphF Fe-S cluster and its histidine ligands are exposed. However, the cluster has a significantly different environment in that five fewer polar groups interact strongly with the cluster sulfide or the cysteinyl ligands. CONCLUSIONS: BphF has structural features consistent with a minimal and perhaps archetypical Rieske protein. Variations in redox potentials among Rieske clusters appear to be largely the result of local electrostatic interactions with protein partial charges. Moreover, it appears that the redox-linked ionizations of the Rieske proteins from proton-translocating complexes are also promoted by these electrostatic interactions.

Molecular basis for the stabilization and inhibition of 2, 3-dihydroxybiphenyl 1,2-dioxygenase by t-butanol.

The steady-state cleavage of catechols by 2,3-dihydroxybiphenyl 1, 2-dioxygenase (DHBD), the extradiol dioxygenase of the biphenyl biodegradation pathway, was investigated using a highly active, anaerobically purified preparation of enzyme. The kinetic data obtained using 2,3-dihydroxybiphenyl (DHB) fit a compulsory order ternary complex mechanism in which substrate inhibition occurs. The Km for dioxygen was 1280 +/- 70 microM, which is at least 2 orders of magnitude higher than that reported for catechol 2,3-dioxygenases. Km and Kd for DHB were 22 +/- 2 and 8 +/- 1 microM, respectively. DHBD was subject to reversible substrate inhibition and mechanism-based inactivation. In air-saturated buffer, the partition ratios of catecholic substrates substituted at C-3 were inversely related to their apparent specificity constants. Small organic molecules that stabilized DHBD most effectively also inhibited the cleavage reaction most strongly. The steady-state kinetic data and crystallographic results suggest that the stabilization and inhibition are due to specific interactions between the organic molecule and the active site of the enzyme. t-Butanol stabilized the enzyme and inhibited the cleavage of DHB in a mixed fashion, consistent with the distinct binding sites occupied by t-butanol in the crystal structures of the substrate-free form of the enzyme and the enzyme-DHB complex. In contrast, crystal structures of complexes with catechol and 3-methylcatechol revealed relationships between the binding of these smaller substrates and t-butanol that are consistent with the observed competitive inhibition.

Purification and preliminary characterization of a serine hydrolase involved in the microbial degradation of polychlorinated biphenyls.

2-Hydroxy-6-oxo-6-phenylhexa-2,4-dienoate (6-phenyl-HODA) hydrolase (BphD), an enzyme of the biphenyl biodegradation pathway encoded by the bphD gene of Burkholderia cepacia LB400, was hyperexpressed and purified to apparent homogeneity. SDS-polyacrylamide gel electrophoresis confirmed that BphD has a subunit molecular mass of 32 kDa, while gel filtration demonstrated that it is a homotetramer of molecular weight 122,000. The enzyme hydrolyzed 6-phenyl-HODA with a kcat of 5.0 (+/- 0.07) s-1 and a kcat/Km of 2.0 (+/- 0.08) x 10(7) M-1 s-1 (100 mM phosphate, pH 7.5, 25 degreesC). The specificity of BphD for other 2-hydroxy-6-oxohexa-2,4-dienoates (HODAs) decreased markedly with the size of the C6 substituent; 6-methyl-HODA, the meta cleavage product of 3-methylcatechol, was hydrolyzed approximately 2300 times less specifically than 6-phenyl-HODA. By comparison, the homologous hydrolase from the toluene degradation pathway, TodF, showed highest specificity for 6-methyl- and 6-ethyl-HODA (kcat/Km of 2.0 (+/- 0.05) x 10(6) M-1 s-1 and 9.0 (+/- 0.5) x 10(6) M-1 s-1, respectively). TodF showed no detectable activity toward 6-phenyl-HODA and 6-tert-butyl-HODA. Neither BphD nor TodF hydrolyzed 5-methyl-HODA efficiently. The kcat of BphD determined by monitoring product formation was about half that determined by monitoring substrate disappearance, suggesting that some uncoupling of substrate utilization and product formation occurs during the enzyme catalyzed reaction. Crystals of BphD were obtained using ammonium sulfate combined with polyethylene glycol 400 as the precipitant. Diffraction was observed to a resolution of at least 1.9 A, and the evaluation of self-rotation functions confirmed 222 (D2) molecular symmetry.

All in the family: structural and evolutionary relationships among three modular proteins with diverse functions and variable assembly.

The crystal structures of three proteins of diverse function and low sequence similarity were analyzed to evaluate structural and evolutionary relationships. The proteins include a bacterial bleomycin resistance protein, a bacterial extradiol dioxygenase, and human glyoxalase I. Structural comparisons, as well as phylogenetic analyses, strongly indicate that the modern family of proteins represented by these structures arose through a rich evolutionary history that includes multiple gene duplication and fusion events. These events appear to be historically shared in some cases, but parallel and historically independent in others. A significant early event is proposed to be the establishment of metal-binding in an oligomeric ancestor prior to the first gene fusion. Variations in the spatial arrangements of homologous modules are observed that are consistent with the structural principles of three-dimensional domain swapping, but in the unusual context of the formation of larger monomers from smaller dimers or tetramers. The comparisons support a general mechanism for metalloprotein evolution that exploits the symmetry of a homooligomeric protein to originate a metal binding site and relies upon the relaxation of symmetry, as enabled by gene duplication, to establish and refine specific functions.

Evolutionary relationships among extradiol dioxygenases.

A structure-validated alignment of 35 extradiol dioxygenase sequences including two-domain and one-domain enzymes was derived. Strictly conserved residues include the metal ion ligands and several catalytically essential active site residues, as well as a number of structurally important residues that are remote from the active site. Phylogenetic analyses based on this alignment indicate that the ancestral extradiol dioxygenase was a one-domain enzyme and that the two-domain enzymes arose from a single genetic duplication event. Subsequent divergence among the two-domain dioxygenases has resulted in several families, two of which are based on substrate preference. In several cases, the two domains of a given enzyme express different phylogenies, suggesting the possibility that such enzymes arose from the recombination of genes encoding different dioxygenases. A phylogeny-based classification system for extradiol dioxygenases is proposed.

Crystal structure of soybean lipoxygenase L-1 at 1.4 A resolution.

Lipoxygenases, which are widely distributed among plant and animal species, are Fe-containing dioxygenases that act on lipids containing (Z,Z)-pentadiene moieties in the synthesis of compounds with a variety of functions. Utilizing an improved strategy of data collection, low temperature, and synchrotron radiation of short wavelength, the structure of ferrous soybean lipoxygenase L-1, a single chain protein of 839 amino acid residues, has been determined by X-ray crystallography to a resolution of 1.4 A. The R-factor for the refined model is 19.7%. General features of the protein structure were found to be consistent with the results of prior crystallographic studies at lower (2.6 A) resolution. In contrast to the prior studies, the binding of a water molecule to the active site Fe was established. The octahedral coordination sphere of the Fe also includes the side chains of His499, His504, His690, and Asn694 as well as the terminal carboxylate of Ile839, which binds as a monodentate ligand. Asn694 is involved in a number of labile polar interactions with other protein groups, including an amide-aromatic hydrogen bond, and appears to be a weak ligand. Several possible access routes for dioxygen and fatty acids to the internal active site and substrate binding cavity are described. The protein structure restricts access to the Fe site such that the formation of an organo-Fe intermediate seems improbable. Structural restrictions pertinent to other proposed reaction intermediates, such as planar pentadienyl and nonplanar allyl radicals, are also discussed.

Crystal structure of the biphenyl-cleaving extradiol dioxygenase from a PCB-degrading pseudomonad.

Polychlorinated biphenyls (PCBs) typify a class of stable aromatic pollutants that are targeted by bioremediation strategies. In the aerobic degradation of biphenyl by bacteria, the key step of ring cleavage is catalyzed by an Fe(II)-dependent extradiol dioxygenase. The crystal structure of 2,3-dihydroxybiphenyl 1,2-dioxygenase from a PCB-degrading strain of Pseudomonas cepacia has been determined at 1.9 angstrom resolution. The monomer comprises amino- and carboxyl-terminal domains. Structural homology between and within the domains reveals evolutionary relationships within the extradiol dioxygenase family. The iron atom has five ligands in square pyramidal geometry: one glutamate and two histidine side chains, and two water molecules.